The long term goal of this project is to develop better therapeutics for NK-LGL leukemia. The proposal represents a new approach to leukemia pathogenesis by positing that imbalances in the sphingolipid rheostat confer the leukemia phenotype. This model postulates that cell fate is determined by the balance between pro- survival (sphingosine-1-phosphate, S1P) and pro-apoptotic (ceramide) lipids. Preliminary data demonstrate altered sphingolipid metabolism in this disease, including increased levels of S1P and decreased levels of ceramide in leukemic LGL;increased expression of acid ceramidase;and findings that stable knockdown of acid ceramidase enhances production of long chain ceramide and results in decreased viability of leukemic LGL. Furthermore, we show that targeting sphingolipid signaling represents a new avenue for therapeutic intervention in this disease. We achieve complete remission of aggressive NK-LGL leukemia by utilizing nanoliposomal formulation of short chain C6 ceramide or the S1P receptor antagonist, FTY720, in the Fischer rat animal model. We will test the hypothesis that S1PR5 signaling mediates survival of leukemic NK cells (Specific Aim 1). Knockdown of S1PR5, the predominant S1P receptor expressed on leukemic NK cells, inhibits constitutive ERK phosphorylation and induces apoptosis. Constitutive overexpression of Rac1, PAK3, RhoB and ROCK1, potential downstream effectors of S1PR5, is observed in leukemic NK cells. Pharmacological inhibition of Gi, RAC1 and ROCK also results in cell death in leukemic NK cells, demonstrating that these targets may be involved in S1PR5-dependent survival pathways. Specific Aim 2 will test the hypothesis that FTY720 induces both apoptosis and autophagy in leukemic NK cells by targeting sphingolipid signaling. We show that pharmacological inhibition of autophagy increases apoptosis induced by FTY720, indicating that blockade of autophagy potentiates FTY720 therapeutic efficacy in leukemic NK cells. FTY720 treatment of leukemic LGL leads to accumulation of intracellular S1P, suggesting that autophagy induced by FTY720 might occur through inhibition of S1P lyase. In addition, we postulate that leukemic NK cells are dependent on survival signaling resulting from NK receptor target recognition in vivo. Convincing preliminary data indicate that sphingolipid survival pathways are activated in normal NK cells after target binding in vitro. Findings similar to those seen in NK-LGL leukemia include upregulation of S1PR5, identification of RhoB, ROCK1, Rac1 and PAK3 as downstream components of S1PR5 signaling, and induction of apoptosis by FTY720. We anticipate that the proposed research will have significant impact on the field of NK cell biology. Our work will define new pathways important for survival of both leukemic and activated normal NK cells. We expect that understanding sphingolipid survival signaling will lead to novel therapeutic approaches for an incurable illness. PUBLIC HEALTH RELEVANCE: This study is investigating leukemia which arises from killer cells of the immune system. The purpose of these studies is to understand the signals which keep the leukemia cells alive. This information can be used to design better treatments.